Experimental evolution of gene essentiality in bacteria

Abstract

AbstractEssential gene products carry out fundamental cellular activities in interaction with other components. However, the lack of essential gene mutants and appropriate methodologies to link essential gene functions with their partners poses significant challenges. Here, we have generated deletion mutants in 32 genes previously identified as essential, with 23 mutants showing extremely slow growth in the SK36 strain ofStreptococcus sanguinis. The 23 genes corresponding to these mutants encode components of diverse pathways, are widely conserved among bacteria, and are essential in many other bacterial species. Whole-genome sequencing of 243 independently evolved populations of these mutants has identified >1000 spontaneous suppressor mutations in experimental evolution. Many of these mutations define new gene and pathway relationships, such as F1Fo-ATPase/V1Vo-ATPase/TrkA1-H1 that were demonstrated across multipleStreptococcusspecies. Patterns of spontaneous mutations occurring in essential gene mutants differed from those found in wildtype. While gene duplications occurred rarely and appeared most often at later stages of evolution, substitutions, deletions, and insertions were prevalent in evolved populations. These essential gene deletion mutants and spontaneous mutations fixed in the mutant populations during evolution establish a foundation for understanding gene essentiality and the interaction of essential genes in networks.SignificanceIdentifying genes that interact with essential genes offers profound theoretical insights into the basic cellular processes of life and practical benefits for fields such as synthetic biology and the fight against drug resistance. Despite its importance, this task is challenging due to the difficulties in creating essential-gene deletion mutants and linking essential gene functions with their interacting partners. In our study, we changed the transformation system to create viable mutants with deletions in genes previously identified as essential inStreptococcus sanguinis. Fortunately, we obtained dozens of essential gene knockout mutants for the first time. These genes, involved in various pathways, are conserved as core components of the essential genome across many bacterial species. Furthermore, through the experimental evolution of 243 independent populations with deletions in these genes, we identified over 1,000 spontaneous suppressor mutations that affect gene essentiality. This collection of spontaneous suppressor mutations, combined with our essential gene deletion mutants, provides a crucial foundation for elucidating gene essentiality and the interactions of essential genes within cellular networks.